ABSTRACT Human somatic cells are exposed to DNA damaging agents on a daily basis from both endogenous and exogenous sources, resulting in a broad range of DNA lesions, varying from DNA breaks and abasic sites to bulky adducts and crosslinks, in normal somatic cells perhaps up to 100,000 lesions per day. Virtually all of that damage is quickly repaired through a complex set of genome maintenance systems, albeit with errors. Such errors result in DNA mutations, which can vary from base substitutions and small deletions or insertions to larger genome structural variation, including chromosomal aberrations. Mutations are true molecular end points, direct indicators of loss of genome sequence integrity. DNA mutations cause cancer and have also been implicated in age- related diseases as well as aging itself. To study mutations in humans, the most widely applied assays are selectable marker assays (e.g., HGPRT), but they can only be applied on cells that can be cultured and cloned, and of course comprise only a very small part of the genome, precluding a more global assessment of mutation loads. With the advent of next-generation sequencing, somatic mutations can be quantitatively assessed, but only in clonal lineages, such as tumors, in which a substantial fraction of cells harbor the same mutations. In normal tissues, somatic mutations are of very low abundance and cannot be distinguished from sequencing errors. Detecting mutations in normal cells and tissues requires either a single cell approach or extremely high accuracy in distinguishing a true mutation from an artifact in sequenced bulk DNA. We recently developed and validated next generation sequencing-based assays for detecting most if not all types of mutations using both bulk DNA and single cell-based approaches. Here we propose to integrate, further optimize and validate these assays into the first next-generation sequencing-based mutation analysis system that provides comprehensive insight in genome sequence integrity in normal human cells. For this proposal, the assay will be tailored to measuring the mutagenic effects of tobacco smoke for testing the hypothesis that mutations in blood or buccal mucosal cells reflect loss of genome integrity in human lung in smokers and non-smokers in relation to lung cancer. In Aim 1, we will develop and validate an integrated and automated assay for measuring the complete landscape of genome instability in epithelial cells exposed in vitro to tobacco smoke condensate. In Aim 2, we will validate the integrated assay for genome sequence integrity in human bronchial, buccal and blood cells exposed in vivo, in relation to tobacco smoke exposure and lung cancer case-control status. The assay developed in the proposed project would potentially allow, for the first time, the use of global genome sequence integrity as an endpoint to assess individual risk of lung cancer in relation to exposure to tobacco smoke, as reflected in non-invasive specimens amenable to epidemiologic studies.